Herpes simplex virus type 2 (HSV-2) and bacterial vaginosis (BV) are two of the most prevalent infections of the female genital tract. Both infections are associated increased risk for HIV acquisition. While HSV-2 and BV have been epidemiologically linked in many studies, the direction and mechanism of their interaction is not known. Our group has pioneered daily participant-collected sampling to show that HSV-2 is detected (shed) frequently, on 10-28% of days, from genital surfaces, and that HSV-2 shedding is asymptomatic the majority of the time. More recent data has demonstrated that HSV-2 genital lesions and asymptomatic shedding episodes are associated with a persistent inflammatory cellular response, with HSV-specific CD4+ and CD8+ T-cells producing IFN-?. These data suggest that HSV-2 infection chronically and fundamentally alters the genital tract. Drs. Marrazzo and Fredricks have similarly used frequent vaginal sampling and molecular methods to document rapid shifts in the vaginal microbiome, and to discover novel BV-associated bacteria (BVAB). This proposal seeks to elucidate the temporal and mechanistic nature of the HSV-2/BV association and to identify the components of the inflammatory response to HSV-2 and BV that may modulate the association. We hypothesize that frequent HSV-2 genital shedding causes increased levels of inflammatory cytokines, such as IL-1 and IFN-?, and decreases in antiviral innate immune molecules (i.e.secretory leukocyte protease inhibitor, SLPI) and that these changes result in fluctuations in the vaginal microbiome which predispose women to develop BV. To address these hypotheses, we will enroll 100 HSV-2 seropositive, HIV seronegative women with a history of recurrent BV and HSV-2, who will self-collect genital and vaginal specimens daily for 28 days. We will quantify HSV using a well-validated PCR assay, and we will measure changes in the microbiome using gram stain (Nugent score), quantitative PCR (qPCR) and broad range 16S rRNA gene PCR with pyrosequencing during HSV shedding episodes. Daily vaginal levels of innate immune proteins and cytokines associated with both HSV-2 and BV will be measured. Relationships between HSV-2 shedding and Nugent score, quantity and presence of BV-associated bacteria (BVAB), and cytokine levels will be determined. In a subset of women, we will determine whether suppression of HSV-2 shedding using acyclovir will result in improved vaginal flora as measured by a decrease in Nugent score. We will also determine whether twice weekly intravaginal metronidazole gel is associated with a decrease in HSV shedding rates. This integrated proposal will determine the relationship between HSV-2 shedding and the vaginal microbiome, and will give insight into the mechanistic basis of the association. Understanding the HSV-2/BV association will provide a framework for appreciating how these infections influence inflammation within the genital tract. Ultimately, this proposal will provide us with knowledge to better promote women's reproductive health.